Transcript Dr. Kathryn Medler
CELL SIGNALING PROCESSES IN TASTE CELLS
KATHRYN MEDLER LAB
Why should we care about taste?
Taste is used to determine if potential food items will be ingested or rejected.
Taste is used by all organisms and is the oldest sensory system.
Loss of taste can lead to depression and loss of appetite which can cause malnutrition. Deficits in the taste system can also lead to uncontrolled appetite and obesity.
Mammalian taste buds are present in papillae on the tongue
Apical Taste stimuli Taste bud Lingual Epithelium Basolateral Afferent gustatory nerve fiber
Taste Transduction: Two distinct signaling pathways exist
Na + H + + + + Bitter Sweet Umami R PLC IP 3 Multiple signaling pathways are present taste cells and all pathways depend on increases in intracellular calcium to transmit signals to the nervous system.
Ca 2+ Store Na + K + [Ca 2+ ] i Serotonin Ca 2+ [Ca 2+ ] i ATP TRPM5 Our lab is studying these different signaling mechanisms: how they function and how they are regulated.
Calcium imaging measures changes in calcium levels in live cells
We can characterize the functional role of different proteins expressed in these cells and how they affect calcium signals.
Using calcium imaging we found:
1750 1500 1250 1000 750 500 250 0 0 “Complex” stimuli Bitter hi K 100 Time (s) 200 300 “Ionic” stimuli 1750 Bitter hi K 1500 1250 1000 750 500 250 0 0 100 200 Time (s) 300 400 Activate calcium release from stores Activate voltage gated calcium influx Different taste stimuli evoke different signals.
Hacker et al., 2008
Surprisingly, we also found
1250 Bitter hi K 1000 750 500 250 0 0 200 Time (s) 400 Some taste cells responded to bitter stimuli AND cell depolarization.
600 Hacker et al., 2008 This is a newly identified sub-population of taste cells.
We asked the question: How do these taste cells respond to multiple stimuli?
Expression patterns of PLC
b
3/IP 3 R1 in taste cells D PLC
b
3 and IP 3 R1 are co-expressed in a population of taste cells that are distinct from the PLC
b
2 expressing cells. Further studies are being conducted to characterize this newly identified signaling pathway.
There are 3 separate taste cell groups.
Na + H + + + + + + + Na + Ca 2+ K + [Ca 2+ ] i
Phospholipase C
b
2 IP 3 R3
IP 3 Ca 2+ + + + Na +
Phospholipase C
b
3 IP 3 R1
IP 3 Ca 2+
Endoplasmic reticulum
[Ca 2+ ] i Ca 2+ We are asking “How do each of these groups contribute to detection of taste stimuli?”
We are also studying the evoked taste responses in obese mice.
We asked “Are peripheral taste responses different in obese mice versus normal mice?” Norm Obese 75 100 Norm Obese NS 75 50 NS 50 25 *** *** 25 *** ** *** *** 0 MPG Sac Ace K Den hi K 0 MPG Sac AceK Den The number of responsive taste cells and the response amplitudes are reduced in obese mice for the appetitive tastes.
We’re asking how does this affect the animal’s ability to perceive taste stimuli? Is it reversible?
We recently identified a new TRP channel in taste cells.
TRPM5 is a well-known monovalent selective TRP channel that is important in taste transduction. TRPM5 turns on in response to some taste stimulation. We found that taste cells also express TRPM4, which is the other monovalent selective TRP channel. TRPM4 is also activated by some taste stimuli.
We are determining the role of TRPM4 in taste transduction.
110 100 90 0 DEN 100 Hi K 200 Time (s) 300
Ca 2+ Na +
20 400 10 0 In some cells, taste stimuli evoke sodium and calcium increases. Using imaging and patch clamp, we’re determining how TRPM4 contributes to these responses.
Gene regulation by WT1 in taste cells
In collaboration with Stefan Roberts lab
Transcriptional Regulation by WT1 BASP1 WT1 General transcription machinery IIA IIF IIH IIB IIE Pol II IID TATA Growth factors Amphiregulin IGFII PDGF-A Apoptosis Bcl 2 Bak c-myc Differentiation Podocalyxin Nephrin
WT1 plays a critical role in the development of several organs and tissues WT1 Knock-out mice Kidneys Gonads Spleen Adrenal glands Diaphragm Retinal Ganglia Olfactory epithelium Taste buds
WT1 and BASP1 are expressed in taste cells Adult Embryonic WT1 WT1 Ctrl BASP1 Ctrl E13 E14.5
E17.5
WT1 null mice fail to develop a peripheral taste system E13.5
WT WT Troma 1 Sox2 KO WT KO WT GAP-43 Shh KO KO
WT1 regulates genes critical for taste cell development Real time PCR CHiP assay
1,2 1 0,8 0,6 0,4 0,2 0
* LEF1
6 5 1 0 4 3 2
LEF1 IgG WT1 BASP1 WT
1,2 1 0,8 0,6 0,4 0,2 0
WT KO * PTCH1
1 0 3 2 5 4 7 6
PTCH1 IgG WT1 BASP1 KO
1,2 1 0,8 0,6 0,4 0,2 0
*
WT1+/+ WT1-/-
BMP4
Primary taste cells can be cultured and transfected Hoechst WT1 Hoechst PLC β2 Hoechst TRMP5-GFP Knockdown of WT1 in cultured taste buds causes a reduction in the expression of WT1 target genes that are important in taste cell maintenance.
4 2 0 14 12 10 8 6 3,5 3 2,5 2 1,5 1 0,5 0
LEF1 IgG PTCH1 IgG CHiP WT1 WT1 BASP1 BASP1 qPCR
1,2 1 0,8 0,6 0,4 0,2 0 1,2 1 0,8 0,6 0,4 0,2 0 1,2 1 0,8 0,6 0,4 0,2 0
WT1 Control siRNA WT1 siRNA LEF1 Control siRNA WT1 siRNA PTCH1 Control siRNA WT1 siRNA
Combine the physiological and molecular approaches of the Medler and Roberts labs to study the role of WT1 and BASP1 in gene regulation during development and tissue homeostasis